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This conference is a major event in the field of Food, Nutrition and Health. It is organized in the frame of the INFOGEST research network(www.cost-infogest.eu) whose objective is to “improve the health properties of food by sharing our knowledge on the digestive process”. INFOGESTis a global network of approximately 582 research scientists (academic and food companies) from 150 institutions across 46 countries.; International audience; Mass spectrometry has become the technique of choice for the simultaneous assessment of a high variety of molecules in complex food matrices. For this reason, it is best suited for precisely monitoring the evolution of digestive processes in vivo and in vitro. However, due to the variety of equipment available in different laboratories as well as settings, statistical evaluations, and interpretations, it is difficult to predict a priori the ideal parametersfor best results. Methods: The present work addressed that gap by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state of the art mass spectrometry applications and analytical capabilities available for studying food digestion.Results: Three representative high-protein foods, skim milk powder, chicken breast and tofu, were digested according to the INFOGEST protocol with sample collection at five time points during gastric and intestinal digestion. Nine laboratories analyzed all digesta with their in house equipment, conventional sample treatment procedure, data evaluation methods, and graphical representation tools. Conclusions: The compiled results demonstrate a strong consensus among labs in terms of major protein degradation for the three foods matrices and present suitable methodical andstatistical approaches for representing the analytical results.

Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines, mitigates the inflammatory response in vitro and in vivo These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e. modulating NF-κB transcription factor activity and IL-8 release) which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and hence the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical and functional properties of EVs produced from cells cultivated on laboratory Yeast Extract Lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant, smaller in diameter and displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacteria interactions.Importance Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in host-microbe interactions. Recently we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems, and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

The role of Propionibacterium freudenreichii in mitigating inflammation has been a subject of study for many years. Lately, it has been shown that immunomodulation properties are strain- specific and that the major responsible for inflammatory modulation ofimmunomodulation by strain CIRM-BIA129 is the presence of the surface layer protein B (SlpB), which has presented immunomodulation even when expressed in other bacteria. Extracellular vesicles (EVs) are nanosized spherical structures, produced by organisms of all kingdoms, including bacteria. They have been associated with inter-organism communication, pathogenesis, competition, and immunomodulation. Recent studies were aiming to address the role of EVs, in the probiotic effects of bacteria. The properties of P. freudenreichii CIRM-BIA129- derived EVs have been investigated. EVs produced by CIRM-BIA129 cultured in milk ultrafiltrate medium (UF) have been characterized by regarding size and morphology. UF-derived EVs displayed a monodisperse pattern with a modal size of 84.80 ± 2.34 nm. They are composed of a wide variety of proteins, mainly involved in metabolic processes, cellular processes and signaling, and storage and processing of information. A, among these proteins, SlpB was found in high abundance. P. freudenreichii EVs were able to inhibit, in a dose dependent manner, the increase of IL-8 production in HT-29 cells induced with LPS, due to NF-KB pathway inhibition, without causing cell cytotoxicity. EVs produced by a CIRM-BIA129 mutant strain with a knockout for SlpB showed a less efficient reduction in IL-8 production. Results have shown that the environmental conditions are able to modify EVs content and, consequently, their immunomodulatory effects. A change in the growth medium, from UF to YEL (yeast extract-lactate) showed a lower production of EVs, with a slightly larger size. EVs produced in YEL did not perform as well in the inhibition of the NF-KB pathway and had no effect on IL-8 production. Recent results have shown that P. freudenreichii EVs were able to protect Caco-2 cells from inflammation-induced excessive increasing permeability. These Altogether, these results show that Evs produced by beneficial propionibacteria are able to generate immunomodulationtrigger immunomodulation, similar to the parental strain. As for the SlpB role in EVs immunomodulation

Les interactions impliquant les bactéries lactiques (BL), responsables des propriétés sanitaires, organoleptiques et santé des produits fermentés, sont souvent étudiées en association avec des levures ou des bactéries propioniques dans divers produits alimentaires fermentés et les mécanismes de leurs interactions sont assez bien caractérisés. En ce qui concerne les interactions entre BL, elles ont principalement été étudiées pour tester des interactions antagonistes. Comprendre comment les BL peuvent interagir positivement serait utile dans de multiples domaines liés à l'alimentation, comme la production de produits fermentés aux propriétés fonctionnelles améliorées ou la fermentation de nouvelles matrices alimentaires. Les dépendances nutritionnelles, notamment celles vis-à-vis des sources d'azote, régissent de nombreuses interactions positives microbiennes. De telles interactions positives ont déjà été étudiées entre les BL du yaourt. Cependant, elles n'ont jamais été exploitées pour créer des co-cultures de BL issues de biotopes différents. L'objectif de cette étude était de favoriser les interactions positives entre BL, basées sur les dépendances aux sources d’azote dans les co-cultures pour augmenter les taux d'acidification, la consommation de sucres comme le raffinose et la production de composés volatils. La stratégie consistait à exploiter les activités protéolytiques et les auxotrophies des acides aminés des BL. Un milieu chimiquement définia été développé pour permettre spécifiquement la croissance des six souches de BL utilisées, trois protéolytiques et trois non protéolytiques. Chacune des souches protéolytiques, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125 et CIRM-BIA244, a été cultivée avec chacune des souches de BL non protéolytiques : L. lactis NCDO2111, Lactiplantibacillus plantarum CIRM-BIA465 et CIRM-BIA1524. La croissancebactérienne a été suivie à l'aide de chambres compartimentées pour comparer la croissance en mono- et co-cultures. Chaque souche protéolytique a induit différents types d'interactions 2 : fortement positives avec E. faecalis CIRM-BIA2412, faiblement positives avec L. lactis NCDO2125, ou nulles avec L. lactis CIRM-BIA244. En co-cultures directes, les interactions ont conduit à des taux d'acidification plus rapides, à un pH final plus bas, à une utilisationplus élevée du raffinose et à des concentrations plus élevées en cinq composés volatils, pouvant donner des arômes intéressants. Les fortes interactions étaient associées à des concentrations plus élevées en tryptophane, valine, phénylalanine, leucine, isoleucine et en peptides. Une analyse peptidomique a permis d’identifier les acides aminés hydrophobes et à chaîne ramifiée comme essentiels dans ces interactions. Cette étude donne un nouvel aperçu des mécanismes régissant les interactions entre BL et offre des solutions pour améliorer ou concevoir des ferments lactiques.

In a general context of food transition, plant-based fermented products experience a considerable development. This results from evolution of consumers’ habits, demanding healthy products with a low environmental impact. This requires research and development, and thus higher scientific knowledge. Fermented soy milks constitute a favorable alternative source of live probiotics. Lactobacillus delbrueckii subsp. bulgaricus and Propionibacterium freudenreichii are extensively used in probiotic dairy products. Their adaptation to the dairy environment has been extensively studied and documented. However, little is known about their adaptation to the soy environment. We investigated suitability of soymilk as a substratefor growth and acidification by Lactobacillus delbrueckii CIRM-BIA1592 and Propionibacterium freudenreichii CIRM-BIA129. A specific focus was made on changes in term of proteome composition, cellular morphology in comparison with the cultivation in cow milk. In order to understand such adaptation, we explored their growth and survival in soy milk ultrafiltrate and compared it to cow’s milk ultrafiltrate. The two species grew in cow milk,yet not in soy milk. P. freudenreichii did grow in soy milk when co-cultured with the lactic acid bacterium L. plantarum while L. delbrueckii did not, even in co-culture with S. thermophilus. Our results revealed major differences in bacterial morphology and proteome composition. Lactobacilli upregulated stress proteins, and downregulated cell cycle and division machinery. Regarding propionibacteria, differential expression of proteins involved in amino acid metabolism, in carbohydrate metabolism, and in stress remediation was observed. These results suggest that changing the fermented substrate may significantly affect both technological and probiotic properties of conventional dairy starters. This should be considered for the development of new fermented functional foods. Screening of a large collection of lactic and propionic acid bacteria is in progress, based on their technological and probiotic properties. Bacterial stains and consortia thereof will be selected for the production of new plant-based fermented functional foods

International audience; Nutritional dependencies, especially those regarding nitrogen sources, govern numerous microbial positive interactions. As for lactic acid bacteria (LAB), responsible for the sanitary, organoleptic, and health properties of most fermented products, such positive interactions have previously been studied between yogurt bacteria. However, they have never been exploited to create artificial co-cultures of LAB that would not necessarily coexist naturally, i.e from different origins. The objective of this study was to promote LAB positive interactions, based on nitrogen dependencies in co-cultures, and to investigate how these interactions affect some functional outputs, e.g. acidification rates, carbohydrate consumption, and volatile compound production. The strategy was to exploit both proteolytic activities and amino acid auxotrophies of LAB. A chemically defined medium was thus developed to specifically allow the growth of six strains used, three proteolytic and three non-proteolytic. Each of the proteolytic strains, Enterococcus faecalis CIRM-BIA2412, Lactococcus lactis NCDO2125, and CIRM-BIA244, was co-cultured with each one of the non-proteolytic LAB strains: L. lactis NCDO2111, Lactiplantibacillus plantarum CIRM-BIA465 and CIRM-BIA1524. Bacterial growth was monitored using compartmented chambers to compare growth in mono- and co-cultures. Acidification, carbohydrate consumption and volatile compound production was evaluated in direct co-cultures. Each proteolytic strain induced different types of interactions: either strongly positive, weakly positive, or no interactions, with E. faecalis CIRM-BIA2412, L. lactis NCDO2125 and L. lactis CIRM-BIA244, respectively. Strong interactions were associated with higher concentrations in tryptophan, valine, phenylalanine, leucine, isoleucine, and peptides. They led to faster acidification rates, lower pH, higher raffinose utilization and concentrations in five volatile compounds. Importance: Lactic acid bacteria (LAB) interactions are often studied in association with yeasts or propionibacteria in various fermented food products and the mechanisms underlying their interactions are being quite well characterized. Concerning interactions between LAB, they have mainly been investigated to test antagonistic interactions. Understanding how they can positively interact could be useful in multiple food-related fields: production of fermented food products with enhanced functional properties or fermentation of new food matrices. This study investigates the exploitation of the proteolytic activity of LAB strains to promote positive interactions between proteolytic and non-proteolytic strains. The results suggest that proteolytic LAB do not equally stimulate non-proteolytic LAB and that the stronger the interactions between LAB are, the more functional outputs we can expect. Thus, this study gives insight into how to create new associations of LAB strains and to guaranty their positive interactions.

International audience; Propionibacterium freudenreichii is a probiotic Gram-positive bacterium with promising immunomodulatory properties. It modulates regulatory cytokines and mitigates the inflammatory response in vitro and in vivo. These properties were initially attributed to specific bacterial surface proteins. Recently, we showed that extracellular vesicles (EVs) produced by P. freudenreichii CIRM-BIA129 mimic the immunomodulatory features of parent cells in vitro (i.e., modulating NF-κB transcription factor activity and interleukin-8 release), which underlies the role of EVs as mediators of the probiotic effects of the bacterium. The modulation of EV properties, and particularly of those with potential therapeutic applications, such as the EVs produced by the probiotic P. freudenreichii, is one of the challenges in the field to achieve efficient yields with the desired optimal functionality. Here, we evaluated whether the culture medium in which the bacteria are grown could be used as a lever to modulate the protein content and, hence, the properties of P. freudenreichii CIRM-BIA129 EVs. The physical, biochemical, and functional properties of EVs produced from cells cultivated on laboratory yeast extract lactate (YEL) medium and cow milk ultrafiltrate (UF) medium were compared. UF-derived EVs were more abundant and smaller in diameter, and they displayed more intense anti-inflammatory activity than YEL-derived EVs. Furthermore, the growth media modulated EV content in terms of both the identities and abundances of their protein cargos, suggesting different patterns of interaction with the host. Proteins involved in amino acid metabolism and central carbon metabolism were modulated, as were the key surface proteins mediating host-propionibacterium interactions. IMPORTANCE Extracellular vesicles (EVs) are cellular membrane-derived nanosized particles that are produced by most cells in all three kingdoms of life. They play a pivotal role in cell-cell communication through their ability to transport bioactive molecules from donor to recipient cells. Bacterial EVs are important factors in hostmicrobe interactions. Recently, we have shown that EVs produced by the probiotic P. freudenreichii exhibited immunomodulatory properties. We evaluate here the impact of environmental conditions, notably culture media, on P. freudenreichii EV production and function. We show that EVs display considerable differences in protein cargo and immunomodulation depending on the culture medium used. This work offers new perspectives for the development of probiotic EV-based molecular delivery systems and reinforces the optimization of growth conditions as a tool to modulate the potential therapeutic applications of EVs.

The article presents a proteomic data set generated by a comparative analysis of the proteomes of Propionibacterium freudenreichii, comparing the CIRM-BIA 129 and CIRM-BIA 1025 strains. The two strains were cultivated until the beginning of the stationary phase in a chemical defined medium (MMO), and in this medium in the presence of NaCl, with or without glycine betaine. Whole-cell proteins were extracted, trypsinolyzed and analyzed by nano LC-MS/MS, prior to identification and classification by function using the X!Tandem pipeline software and the proteomic data from NCBI.nlm.nigh.gov. Quantification of proteins was then carried out in order to detect change in their expression depending on the culture medium. This article is related to the research article entitled “Benefits and drawbacks of osmotic adjustment in Propionibacterium freudenreichii”. The comparative proteomic analysis of the two strains reveal strain-dependent and medium-dependent stress proteomes in the probiotic P. freudenreichii.

International audience

IntroductionIn order to minimize the denaturation of bioactive compounds, which is known to affect donor humanmilk (HM) after the mandatory heat treatment (Holder pasteurization at 62.5°C, 30min – HoP), anumber of alternative processing techniques are currently being investigated. Recently, High Temperature-Short Time pasteurization (HTST, 72°C-15s) was found to limit the denaturation of somebioactive components of human milk.ObjectiveThe aim of the study was to investigate whether different types of pasteurization (HoP and HTST)may differently affect peptide release from human milk during preterm infant gastrointestinal digestion,by using a dynamic in vitro systemMethodologyPooled raw HM (RHM) from 5 donors was collected and processed in triplicate according to HoP andHTST. The pasteurized milks and RHM were digested in triplicate using a dynamic in vitro digestionsystem, mimicking the preterm physiology conditions. Samples were collected at different digestiontimes. Peptides from undigested and digested samples were analysed using a Q-Exactive massspectrometer and peptide abundance was subjected to multivariate statistics to unravel specificprofile trends.Main findingsPre-proteolysis occurred mostly on β-casein, from which originated more than 82% of the peptidesfound in undigested milk. During digestion, a differential behaviour between gastric and intestinalpeptide release was found by multivariate statistics. In particular, whereas in the gastric phase thepeptide pattern was more similar for HTST and RHM with respect to HoP, the opposite was found atthe beginning of the intestinal phase, while at the end of digestion no difference was found. Thesedifferences were mostly due to peptides released from β-casein, especially in the gastric phase.Some bioactive peptides from bile salt-stimulated lipase, caseins and lactoferrin presented significantlydifferent abundances between the different samples during intestinal digestion.ConclusionThe results indicate possible consequences of the different pasteurizations on the biological activityof donor’s human milk.

Dans les pays en développement, quels que soient le climat et lesespèces animales domestiquées, le lait et ses dérivés occupent leplus souvent une place importante dans la vie quotidienne et constituentun vecteur indéniable de développement. La diversité des pratiques,des outils et des produits nis est considérable et implique unensemble d’acteurs le long des chaînes d’approvisionnement. Parfoismenacés par les dynamiques en cours de la mondialisation, le lait etses dérivés représentent néanmoins une valeur patrimoniale inestimable.Outre leur rôle nutritif incontesté, ces produits ont souvent unrôle clé dans la microéconomie et le développement territorial, avecde multiples modèles selon les contraintes et les opportunités locales.L’objectif de ces rencontres, qui font suite à la 1re édition organisée àRennes (France) en 2014 (colloque6.inra.fr/lait2014), est de favoriser leséchanges d’expériences autour de la diversité des modèles de développementlaitier observés à l’échelle de la planète, de rassembler toutes lesinitiatives et réseaux impliqués, de mesurer l’importance des femmes etdes jeunes dans ces dynamiques et de m@ieux cerner le rôle des politiquespubliques pour accompagner cet essor.Aboutir ensemble à une meilleure coordination des actions autour dulait dans les pays en voie de développement, et notamment à des projetsconjoints de recherche, de formation ou d’appui est la principaleambition de ces rencontres.; La chymosine est l’enzyme communément utilisée pour coaguler le lait en fromagerie mais des coagulants d’origine microbienne et végétale existent aussi. Ces derniers présentent l’avantage d’être accessible et facile d’utilisation même à l’échelle artisanale.En Afrique de l’Ouest, les feuilles et tiges de l’arbuste Calotropis procera sont utilisées dans la fabrication d’un fromage traditionnel appelé « wagachi » qui signifie en langue peulh « gâteau de lait ».Ce fromage est utilisé en remplacement de la viande ou du poisson dans nombre de mets traditionnels. L’importance de sa consommation incite à mener des travaux dans le but d’améliorer la qualité du produit.

Chymosin is the major enzyme used in cheesemaking but latex enzymes are also used. The aim of this work was to characterize the composition and the structure of dairy gel obtained by an extract of Calotropis procera leaves in comparison with those obtained by chymosin. The biochemical and mineral compositions of the curds and the cheese yields obtained by using Calotropis procera extract or chymosin were relatively similar. Quantitative and qualitative evaluations of proteolysis after milk coagulation, determined by the non-protein nitrogen content and chromatography coupled to mass spectrometry, indicated that Calotropis procera extract was more proteolytic than chymosin and that κ-casein was proteolyzed. The main consequence of proteolysis by Calotropis procera extract or chymosin was the formation of a similar and regular network with the presence of aggregates of casein micelles. These results support that Calotropis procera extract can be used as effective coagulant in cheesemaking.

Dans les pays en développement, quels que soient le climat et lesespèces animales domestiquées, le lait et ses dérivés occupent leplus souvent une place importante dans la vie quotidienne et constituentun vecteur indéniable de développement. La diversité des pratiques,des outils et des produits nis est considérable et implique unensemble d’acteurs le long des chaînes d’approvisionnement. Parfoismenacés par les dynamiques en cours de la mondialisation, le lait etses dérivés représentent néanmoins une valeur patrimoniale inestimable.Outre leur rôle nutritif incontesté, ces produits ont souvent unrôle clé dans la microéconomie et le développement territorial, avecde multiples modèles selon les contraintes et les opportunités locales.L’objectif de ces rencontres, qui font suite à la 1re édition organisée àRennes (France) en 2014 (colloque6.inra.fr/lait2014), est de favoriser leséchanges d’expériences autour de la diversité des modèles de développementlaitier observés à l’échelle de la planète, de rassembler toutes lesinitiatives et réseaux impliqués, de mesurer l’importance des femmes etdes jeunes dans ces dynamiques et de m@ieux cerner le rôle des politiquespubliques pour accompagner cet essor.Aboutir ensemble à une meilleure coordination des actions autour dulait dans les pays en voie de développement, et notamment à des projetsconjoints de recherche, de formation ou d’appui est la principaleambition de ces rencontres.; Chymosin is the common enzyme used in cheese making worldwide but other enzymes especially from vegetable origin also exist. In West Africa, an extract of the shrub Calotropis procera, is traditionally used to make a local soft cheese named Wagashi. This cheese, produced and marketed by women, is an interesting source of incomes. The aim of this study was to understand the milk coagulation mechanism by an extract of Calotropis procera leaves in comparison with chymosin. A standard manufacturing process was set up for making both Calotropis procera and chymosin curds. The contents in dry matter, ash and total nitrogen were more important for the curds manufactured using Calotropis procera (310,9; 31,1 and 231,9 g.Kg-1, respectively) compared to those made using chymosin ( 305,7; 25,5 and 207,8 g.Kg-1, respectively). The whey liquid dry matter was also more important when Calotropis procera extract was used. In spite of these composition differences considered as small, both types of curds can be classified as rennet curds strongly mineralized. Analysis by chromatography coupled to tandem mass spectrometry showed an increasing number of peptides coming from -caseins hydrolysis identified in the whey liquids: 96 when using Calotropis procera extract and 47 when using chymosin as coagulant. The proteolysis of this casein molecule explains the milk coagulation observed in both cases. The observation of the two curds by scanning electron microscopy have revealed regular networks with the presence of aggregates of casein micelles. These results support that Calotropis procera extract can be used as effective coagulant in cheese manufacturing with technological adaptations to take in consideration and specific organoleptic and textural specifies.

Propionibacterium freudenreichii is widely used as cheese ripening starter. Its probiotic effects have also been reported at the gut level, including modulation of bifidobacteria, of colon epithelial cells proliferation and apoptosis and of intestinal inflammation. They rely on active metabolism in situ. Growth media and growth conditions determine stress tolerance acquisition in P. freudenreichii, thereby determining its probiotic efficacy in the final products. A simplified process from growth to spray drying of propionibacteria was developed using hyperconcentrated sweet whey as a 2-in-1 medium to both culture and protect P. freudenreichii from spray drying, without harvesting and washing step. The hypertonic stress in hyperconcentrated sweet whey led to overexpression of key stress proteins, accumulation of intracellular storage molecules, compatible solutes, enhanced multistress tolerance acquisition, as well as enhanced survival upon spray-drying. This work opens new perspectives for the sustainable development of new starter and probiotic preparations with enhanced robustness.